Lingchen Fu

BAFF-R promotes cell proliferation and survival through interaction with IKKβ and NF-κB/c-Rel in the nucleus of normal and neoplastic B-lymphoid cells

BLyS and its major receptor BAFF-R have been shown to be critical for development and homeostasis of normal B lymphocytes, and for cell growth and survival of neoplastic B lymphocytes, but the biologic mechanisms of this ligand/receptor-derived intracellular signaling pathway(s) have not been completely defined. We have discovered that the BAFF-R protein was present in the cell nucleus, in addition to its integral presence in the plasma membrane and cytoplasm, in both normal and neoplastic B cells. BAFF-R interacted with histone H3 and IKKbeta in the cell nucleus, enhancing histone H3 phosphorylation through IKKbeta. Nuclear BAFF-R was also associated with NF-kappaB/c-Rel and bound to NF-kappaB targeted promoters including BLyS, CD154, Bcl-xL, IL-8, and Bfl-1/A1, promoting the transcription of these genes. These observations suggested that in addition to activating NF-kappaB pathways in the plasma membrane, BAFF-R also promotes normal B-cell and B-cell non-Hodgkin lymphoma (NHL-B) survival and proliferation by functioning as a transcriptional regulator through a chromatin remodeling mechanism(s) and NF-kappaB association. Our studies provide an expanded conceptual view of the BAFF-R signaling, which should contribute a better understanding of the physiologic mechanisms involved in normal B-cell survival and growth, as well as in the pathophysiology of aggressive B-cell malignancies and autoimmune diseases.

Nuclear Localization in the Biology of the CD40 Receptor in Normal and Neoplastic Human B Lymphocytes

CD40 is a tumor necrosis factor (TNF) receptor superfamily, (TNFR; TNFRSF-5) member, that initiates important signaling pathways mediating cell growth, survival, and differentiation in B-lymphocytes. Although CD40 has been extensively studied as a plasma membrane-associated growth factor receptor, we demonstrate here that CD40 is present not only in the plasma membrane and cytoplasm but also in the nucleus of normal and neoplastic B-lymphoid cells. Confocal microscopy showed that transfected CD40-green fluorescent fusion protein entered B-cell nuclei. The CD40 protein contains a nuclear localization signal sequence that, when mutated, blocks entry of CD40 into the nucleus through the classic karyopherins (importins-α/β) pathway. Nuclear fractionation studies revealed the presence of CD40 protein in the nucleoplasm fraction of activated B cells, and chromatin immunoprecipitation assays demonstrated that CD40 binds to and stimulates the BLyS/BAFF promoter, another TNF family member (TNFSF-13B) involved in cell survival in the B cell lineage. Like other nuclear growth factor receptors, CD40 appears to be a transcriptional regulator and is likely to play a larger and more complex role than previously demonstrated in regulating essential growth and survival pathways in B-lymphocytes.

Constitutive BR3 receptor signaling in diffuse, large B-cell lymphomas stabilizes nuclear factor-κB-inducing kinase while activating both canonical and alternative nuclear factor-κB pathways.

Aberrant nuclear factor κB (NF-κB) signaling has been found to be of particular importance in diffuse, large B-cell lymphoma (DLBCL) cell survival and proliferation. Although the canonical NF-κB signaling pathway has been studied in some detail, activation of the alternative NF-κB pathway in DLBCL is not well characterized. Important insights into the regulation of the alternative NF-κB pathway in B lymphocytes has recently revealed the regulatory importance of the survival kinase NIK (NF-κB-inducing kinase) in genetically engineered murine models. Our studies demonstrate that both the canonical and alternative NF-κB pathways are constitutively activated in DLBCL. We also demonstrate that NIK kinase aberrantly accumulates in DLBCL cells due to constitutive activation of B-cell activation factor (BAFF)-R (BR3) through interaction with autochthonous B-lymphocyte stimulator (BLyS) ligand in DLBCL cells. Activation of BR3 in DLBCL induces recruitment and degradation of tumor necrosis factor receptor-associated factor 3, which results in NIK kinase accumulation, IκBα phosphorylation, and NF-κB p100 processing, thereby resulting in continuous activation of both NF-κB pathways in DLBCL cells, leading to autonomous lymphoma cell growth and survival. These results further elucidate mechanisms involved in abnormal NF-κB activation in DLBCL, and should contribute to better future therapeutic approaches for patients with DLBCL.

Nuclear Tumor Necrosis Factor Receptor-associated Factor 6 in Lymphoid Cells Negatively Regulates c-Myb-mediated Transactivation through Small Ubiquitin-related Modifier-1 Modification

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-κB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5′-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.

BLyS/BR3 Receptor Signaling in the Biology and Pathophysiology of Aggressive B-Cell Lymphomas

Lymphoid neoplasms usually display various genetic alterations that dysregulate normal immune functions, primarily through abnormal cellular growth and survival mechanisms that expand and immortalize a single neoplastic clone, to the detriment of the indigenous normal immune system, and eventually the host. In addition to the nonrandom genetic translocations, other important cellular abnormalities are also involved. The abnormalities in cellular regulation generally occur in one or more of the following four areas: cell death, cell cycle progression, NF-κB activation, or critical signal transduction pathways, usually from cell surface growth and survival receptors. Recent studies of BLyS/BAFF family members, BLyS and APRIL, have shown their importance in non-Hodgkin’s B-cell lymphoma (NHL-B), chronic lymphoid leukemia (CLL), and plasma cell myeloma cell growth and survival. In this chapter, we will discuss how BLyS is involved in mechanisms of tumor cell proliferation and survival in these malignant lymphoid cells. Potential therapeutic approaches to these lymphoid malignances through inhibition of BLyS signaling pathways are also discussed.